Intestinal toxicity produced by anti-folate treatment with amethopterin has seriously limited the use of this drug in chemotherapy of cancer and other diseases. The toxicity is thought to be related to inhibition of dihydrofolate reductase by amethopterin, which results in a block of key metabolic pathways and death of intestinal cells. The mechanism of amethopterin-dihydrofolate reductase interaction, as well as the membrane transport of amethopterin (and other folate compounds), have been under study by the applicant using in vitro preparations from rat intestine. These techniques and others described in the text will be applied to an understanding of: 1) the interaction of amethopterin with dihydrofolate reductase and the ways in which this interaction can be minimized to reduce the toxic effect; 2) the mechanisms of transport of amethopterin and other folate compounds in the intestine and how transport may be controlled; and 3) the properties of intestinal dihydrofolate reductase and how these properties contrast in normal and malignant states, and how they may be utilized in the diagnosis and treatment of malignant disease.